The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of crosslinking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.Over the past decade it has been established that in both higher eukaryotes and yeast, the enzyme oligosaccharyl transferase (OT), 1 is a complex consisting of multiple, nonidentical membrane protein subunits residing in the endoplasmic reticulum (ER) (1). In the case of the yeast Saccharomyces cerevisiae, nine different subunits have been cloned and identified. Among them, the genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; OST4 gene is essential for growth of the cell at 37°C, but not at 25°C; Ost3p, Ost5p, Ost6p subunits are not essential for the viability of the cell, but are required for maximal activity of the enzyme complex in catalyzing N-glycosylation (for review, see Refs. 1 and 2). To understand how these nine subunits interact with each other, a genetic approach using the yeast two-hybrid screen was utilized, but it failed to show interactions between any of the luminal domains of the yeast OT subunits (3). Studies on the mechanism of enzyme catalysis have proposed that Wbp1p may contain a site for the binding of the lipidlinked oligosaccharide substrate (4), and Stt3p was shown to be directly involved in peptide substrate recognition and/or the catalytic glycosylation process (5). However, there is little detailed information on how these two subunits interact to catalyze the glycosylation reaction.Study of the structural interactions of membrane proteins of the ER offers a special challenge because of the hydrophobicity of the proteins and the special environ...

Section: Four Essential Gene Products Namely Ost1p Wbp1p Swp1p Ansupporting

confidence: 76%

Section: Four Essential Gene Products Namely Ost1p Wbp1p Swp1p Anmentioning

confidence: 87%

New Findings on Interactions among the Yeast Oligosaccharyl Transferase Subunits Using a Chemical Cross-linker

Yan

Ahmed

Qi³

et al. 2003

“…The reaction was stopped by addition of chloroform/methanol to give a ratio of chloroform/ methanol/water of 2:1:1 (by volume) and processed further by phase separation (28) using an upper phase of chloroform/methanol/water of 1:32:48 (by volume) and collecting both lower and interphase. The (␣1-6)-linkage in the Man(␣1-6)Man(␤1-4)GlcNAc 2 -PP-dolichol product formed was determined by incubation of the tetrasaccharide, released from the lipid by mild acid hydrolysis (30), with recombinant ␣1-6-mannosidase (Calbiochem) or recombinant ␣1-3-mannosidase (Calbiochem) and HPLC analysis. Whereas in the case of ␣1-6-mannosidase treatment (1.1 milliunits of enzyme, 3.5 h of incubation), Man(␣1-6)Man(␤1-4)GlcNAc 2 was converted to Man(␤1-4)GlcNAc 2 , and no digestion was observed using ␣1-2,3-mannosidase (10 units of enzyme, 24 h of incubation).…”

Section: Preparation Of Man(␣1-6)man(␤1-4)glcnac 2 -Pp-dolichol-man(␣1-mentioning

confidence: 99%

“…Man 5 -LLO was isolated from ⌬alg3 yeast cells metabolically labeled with [2-3 H]mannose as described previously (30). Man 5 -LLO (18,000 cpm) was dispersed by sonication in 100 mM sodium acetate, pH 5.0, containing 2 mM ZnCl 2 and incubated with 3.75 milliunits of enzyme at 37°C for 50 min under shaking in a final volume of 0.015 ml.…”

Section: Preparation Of Man(␣1-6)man(␤1-4)glcnac 2 -Pp-dolichol-man(␣1-mentioning

confidence: 99%

A New Type of Congenital Disorders of Glycosylation (CDG-Ii) Provides New Insights into the Early Steps of Dolichol-linked Oligosaccharide Biosynthesis

Thiel

Schwarz

Peng

et al. 2003

Self Cite

114

Deficiency of GDP-Man:Man 1 GlcNAc 2 -PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man 1 GlcNAc 2 -PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man 1 GlcNAc 2 -PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an ␣1,3-mannosyltransferase. The resulting Man␣1,3-ManGlcNAc 2 -PP dolichol is further elongated by a yet unknown ␣1,6-mannosyltransferase.Congenital disorders of glycosylation (CDG) 1 compose a rapidly growing group of inherited multisystemic disorders in man, which are commonly associated with severe psychomotor and mental retardation (1). The characteristic biochemical feature of CDG is defective glycosylation of proteins due to mutations in genes required for the biosynthesis of N-linked oligosaccharides.The attachment of oligosaccharide chains onto newly synthesized proteins is one of the most widespread forms of co-and post-translational modifications and is found in animals, plants, and bacteria. Glycoproteins are located inside cells predominantly in subcellular organelles and in cellular membranes and most abundantly in extracellular fluids and matrices. The oligosaccharide moiety of the glycoproteins can affect their folding, their transport, as well as their biological activity and stability (2, 3). The complex process of protein glycosylation requires more than a hundred glycosyltransferases, glycosidases, and transport proteins. CDG are classified into two groups. Defects of the assembly of lipid-linked oligosaccharides or their transfer onto nascent glycoproteins compose CDG type I, whereas CDG type II includes all defects of trimming and elongation of N-linked oligosaccharides (4). In the past 7 years the molecular nature of eight CDG-I and four CDG-II types could be identified (5-24).Here we describe for the first time a molecular defect in glycoprotein biosynthesis in man which affects at the cytosolic side of the endoplasmic reticulum the transfer of mannosyl residues from GDP-Man to Man 1 Glc...

“…Transformation into yeast and E. coli was carried out using standard techniques. (25). The cells were grown overnight at 25°C.…”

mentioning

confidence: 99%

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

Kämpf

Absmanner

Schwarz

et al. 2009

Self Cite

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the ␣1,3-and the ␣1,6-mannose-linked residue from GDP-mannose to Man 1 GlcNAc 2 -PP-Dol forming the Man 3 GlcNAc 2 -PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N-or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX 7 E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys 230 , as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1-4), and several features of this pathway even occur in prokaryotes (5-7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8 -10). The initial steps of the dolichol pathway up to Man 5 GlcNAc 2 -PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER), 2 using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14 -17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18 -20).One of the tempe...